The present invention relates to the detection and identification of infectious diseases. Specifically, the present invention relates to a method and apparatus for the detection of disease-causing particles such as bacteria, viruses and other particulate entities in liquid samples with extremely high sensitivity.
Quartz crystal microbalances (xe2x80x9cQCMxe2x80x9d) have been developed as sensitive chemical and biochemical sensing devices and can be used for the detection of disease-related particles such as viruses and bacteria in liquid samples (see e.g. Thompson, M. et al., Analyst Vol. 116, pp. 881-890, 1991; Rickert, J. et al, Biosensors and Bioelectronics Vol. 12, pp. 567-575, 1997; Uttenthaler, E. et al., Biosensors and Bioelectronics 16, 735-743, 2001). In this technology, a binding partner such as an antibody is attached to the surface of a small resonant quartz crystal with a mechanical resonance frequency typically in the 10 to 30 MHz region. If a disease-related particle binds to the antibody, the resonance frequency of the quartz crystal shows a very small shift, whereby such shift in frequency or a correlated phase shift between the electrical excitation and the mechanical vibration is an indication that an antibody-specific binding partner was present in the liquid sample.
A significant improvement in the detection sensitivity of a QCM biosensor has been achieved by applying the technology of rupture event scanning (xe2x80x9cREVSxe2x80x9d), (see Dultsev, F. N. et al., Langmuir Vol. 16, 5036-5040, 2000; Cooper, M. A. et al., Nature Biotechnology Vol. 19, 833-837, 2001; WO 01/02857 A1 to Klenerman et al.). In the REVS technology, as in the classic QCM technology, a binding partner such as an antibody is attached to the surface of a small resonant quartz crystal with a mechanical resonance frequency typically in the 10 to 20 MHz region. The liquid sample containing bacteria or viruses is brought into contact with the activated crystal surface so that binding events can take place.
After a 30-minute incubation period, the resonant quartz crystal is operated as close as possible to the fundamental mechanical resonance frequency, whereby the driving power for the quartz crystal is monotonously increased, until suddenly the binding between the binding partners is broken up. According to the inventors of REVS, the breaking or xe2x80x9crupturexe2x80x9d event can be detected due to the emission of noisy sound waves with a preferred frequency spectrum around the third harmonic of the fundamental resonance frequency. The quartz crystal acts as a sensitive microphone, and the generated electrical signal is monitored via an electric resonance circuit tuned to a frequency close to the third harmonic of the fundamental resonance frequency of the crystal. The REVS technology has the potential of detecting the breaking-away of only a few binding partners, thereby enabling extremely sensitive detection.
As mentioned above, a certain incubation time is required to bring the targets such as bacteria or viruses that are present within the liquid sample into contact with the activated crystal surface, so that binding events can take place. Movement of the targets usually takes place due to diffusion. The time, t, needed for a target to cross over a distance, d, via diffusion is given by the equation t=d2/2D, where D is the so-called diffusion coefficient. Assuming a typical diffusion coefficient D=7*10 Cm2/s for a large molecule, such target would need two hours to cross over a distance of 1 mm. Even larger distances may be required if the sample volume cannot be extremely small, which is often the case for medical samples having a low target concentration.
One could try to resolve this problem by pre-concentrating the target molecules via centrifugation or similar process steps, but this requires significant extra effort and makes the detection more time-consuming and more expensive. One goal of the present invention is, therefore, to avoid the need for a pre-concentration step. Other goals of the invention are to allow for the handling of large sample volumes, and to allow for multiplexed detection within one detection device.
The present invention is related to diagnostic sensing devices where a liquid sample has to come in contact with a sensing surface, or with a plurality of sensing surfaces. The invention is in particular of interest for sensing devices where the sample liquid contains the target molecules that have to be detected in low concentration, and where, therefore, a large sample volume has to be processed to achieve detection at all. The present invention avoids the need for a pre-concentration step, and also allows for the handling of large sample volumes, and to further increase sensitivity through multiplexed detection within one detection device.